The overall goal of this grant is to understand why polyamines are essential to the migration of GI mucosal cells during the early mucosal restitution component of healing. Experiments supported during the past 20 years this project has been active have shown that 1) polyamines are essential to the healing of gastric and duodenal mucosa, 2) polyamines are required for mucosal restitution which depends on cell migration, 3) polyamines are essential to cell migration, 4) polyamines are required for integrin signaling and focal adhesion complex formation, 5) Rac activity is both essential and sufficient for cell migration in IEC-6 cells depleted of polyamines, 6) Src-kinase is constitutively active in polyamine depleted cells, and 7) focal adhesion kinase is inhibited in polyamine depleted cells. Thus, we now know that polyamines influence the signaling cascade that leads to migration of GI epithelial cells at some point(s) between the cell membrane and Rac. We also know that the formation of focal adhesion complexes and Src activity are likely to be involved. The overall goal of the studies in the current application is to elucidate how and where polyamines interact to influence the cascade that regulates the activation of Rac. The first set of specific aims will determine the role of focal adhesion assembly on the activation of Rac. Preliminary data indicate that the inhibition of focal adhesion kinase (FAK) decreases the activity of Rac, and that it does so by preventing the activation of the GEFs responsible for activating Rac. Activation of GEFs using constitutively active constructs also increased cell migration and Rac activity. The second major group of aims will determine the role of Src in the formation of focal adhesion complexes. Preliminary data indicate that in normal cells Src is responsible for activating FAK and the subsequent formation of focal adhesions, and in polyamine depleted cells there is a decreased association between Src and FAK. The third specific aim seeks to define mechanistically how polyamines modulate the function of Src. Using specific monoclonal antibodies to Src and spermine, we have evidence that polyamines bind Src on its SH2 domain, the domain responsible for binding FAK. We will then test whether polyamines must be present for Src to bind FAK. These studies will provide the first explanation of an effect of polyamines at the molecular level on a signaling cascade and the physiological process that it regulates. This work will increase our understanding of cell function basic to many clinical conditions in which cell migration is a major component. These include peptic ulcer disease, cancer and inflammatory bowel disease.